Protease purification

Sumo Protease Expression and Purification

SUMO-fusion protein purification system.

This is a protein purification system similar to one distributed by Invitrogen and based on the work of Christopher Lima (see Mol Cell (2000) 5:865-876).

Since Invitrogen charges way too much money for the reagents, I decided to construct my own system.

Overview

This system will allow you to overexpress and purify a SUMO-fusion protein and SUMO protease both as 6xHis tagged proteins. The 6xHis-SUMO tag can then be cleaved from your favorite protein with the protease and, since both the cleaved tag and protease have 6xHis tags, they can be removed from the protein prep with Ni-NTA resin.

How the constructs were made

To make the system I amplified a DNA fragment encoding an active Ulp1 (SUMO-protease) fragment [Ulp1(403-621)] from yeast genomic DNA. I then cloned it as an N-terminal 6xHis fusion in a pET-derived vector for expression from the PT7 promoter. The resulting construct is called pTB145 [PT7::H-Ulp1(403-621)].

For SUMO fusions, I amplified Smt3 (SUMO) from yeast genomic DNA and cloned it into a pET-derived expression vector also as an N-terminal 6xHis fusion. The resulting construct is called pTB146 [PT7::H-SUMO]. It was cloned so that precise gene fusions to SUMO can be constructed with NO ADDED sequences. You can then make you SUMO-protein fusions and cleave off the SUMO portion without leaving any additional non-native amino acids on your protein.

Plasmid sent

pTB145 - for overexpressing the SUMO protease

*IMPORTANT NOTE* SUMO protease will not cleave if your protein starts with Pro, Leu, Lys, or Val as the first amino acid after the GlyGly cleavage site.

Purifying H-Ulp1 (403-621)

My protocol:

Expression and Purification of UlpI

Ulp1 Expression:

20ml LB (containing 100ug/ml ampicillin) was in inoculated with a single colony of E. coli BL21(DE3) containing pTB146-UlpI and grown overnight at 37C at 200rpm. 2L of LB (containing 100ug/ml ampicillin) was inoculated with 20ml of overnight culture, incubated at 37C & 200rpm for 3h (O.D.600 = 0.5) and then induced with 0.25mM IPTG followed by growth at 16C, 200rpm for overnight. Cells were harvested by centrifugation at 4100rpm for 10min and cell pellet was stored at -80C.

Cell Lysis & Ulp1 Purification:

Cells were thawed and resuspended in 80ml of Buffer UA (50mM NaPi (pH 8.0), 300mM NaCl, 10mM Imidazole) and lysed using EmulsiFlex (Avestin). Centrifuged the cell lysate at 20,000rpm for 60min. Loaded the supernatant onto 1ml Ni-NTA agarose resin equilibrated with Buffer UA and collected the flow through. Washed the column with 10 column volumes of Buffer UA followed by washing with 5ml of 20mM of Imidazole twice. Eluted the protein with 5ml of 30, 60, 110, 210 and 510mM of Imidazole in Buffer UA. Proteins were subjected to SDS-PAGE analysis.

Fractions containing UlpI were loaded onto Source -Q equilibrated with Buffer UB (50mM NaPi (pH 8.0), 50mM NaCl) and the UlpI came out in flow-through which was collected and concentrated using 10kDa cutoff ultrafiltration and analyzed by SDS –PAGE. Protein was dialyzed against storage buffer U (25mM Tris (pH 8.0), 200mM NaCl; 50% glycerol; 0.5% NP-40; 0.5mM DTT).

The prep was then aliquoted, snap frozen, and stored at -80oC.

Once thawed, aliquots can be kept at -20oC like commercial enzymes. They remain active for at least a couple of weeks if not longer.

Cleavage

I perform the cleavage on ice overnight at a 100:1 ratio of SUMO fusion to protease. Titration experiments were done to determine the optimal ratio and 100:1 worked well.

Sample reaction

500ul H-SUMO-ProteinX (819uM)

27ul H-Ulp1(403-621) (148uM)

160ul 5x cleavage buffer

273ul H2O

5X Reaction buffer -(250mm Tris-HCl (pH 8.0), 1% NP40, 750mM NaCl, 5mM DTT)

To remove the H-SUMO tag and protease I add 100ul of NiNTA resin to the reaction after it was incubated overnight on ice. Rotate for 1-2hrs at 4oC, pellet the resin, and carefully remove the supernatant (don't be greedy).

Now you have untagged native protein that's reasonably pure and can be tested for activity or purified over another column.

Alternative Purification Protocol:

Grow a 8ml overnight culture of BL21(lDE3)/plysE/pTB145 in LB-Amp(50ug/ml)-Cam(35ug/ml)-Glucose(0.1%) at 37oC

Add to 2L of LB-Amp and grow at 37oC to an OD600=0.8

Add IPTG to 0.84mM, Glucose to 0.04% and continue growing for 6hrs at 25C.

Pellet cells, and store at -80oC.

To break cells. Thaw at 37oC, snap freeze, thaw at 37oC, then place on ice. Sonicate 3-4 times for 30 sec for each 10ml aliquot. Keep on ice at all times. Check breakage under the microscope (100x phase). If better breakage is needed, sonicate a few more times.

Pellet extracts at 200,000xg for 60min.

Save aliquot of supernatant for gel (50ul is plenty) and put the rest over a 0.5ml column of NiNTA resin from Qiagen. Keep at 4oC in beer cooler or coldroom.

Collect flowthrough

Wash column 4x1ml Column buffer (20mM imidazole)

Start step elution: 2x0.25ml Column buffer (50mM imadazole)

3x0.25ml Column buffer (100mM imadazole)

3x0.25ml Column buffer (250mM imadazole)

2x0.25ml Column buffer (500mM imidazole)

Run 12% SDS-PAGE gels with all fractions (2.5ul each plus 2.5ul 2x sample buffer is plenty for mini-gels. Stain with Coomassie to see where H-Ulp1(403-621) elutes.

Mine came out in fractions elution fractions 5&6. I pooled them and dialyzed against 50mM Tris(pH 8.0), 200mM NaCl, 10% glycerol and 1mM DTT.

I then adjusted the solution conditions to: 25mM Tris (pH 8.0), 200mM NaCl, 50% glycerol, 0.5% NP-40, and 0.5mM DTT.

Sumo System

SUMO-fusion protein purification system.

This is a protein purification system similar to one distributed by Invitrogen and based on the work of Christopher Lima (see Mol Cell (2000) 5:865-876).

Since Invitrogen charges way too much money for the reagents, I decided to construct my own system.

Overview

This system will allow you to overexpress and purify a SUMO-fusion protein and SUMO protease both as 6xHis tagged proteins. The 6xHis-SUMO tag can then be cleaved from your favorite protein with the protease and, since both the cleaved tag and protease have 6xHis tags, they can be removed from the protein prep with Ni-NTA resin.

How the constructs were made

To make the system I amplified a DNA fragment encoding an active Ulp1 (SUMO-protease) fragment [Ulp1(403-621)] from yeast genomic DNA. I then cloned it as an N-terminal 6xHis fusion in a pET-derived vector for expression from the PT7 promoter. The resulting construct is called pTB145 [PT7::H-Ulp1(403-621)].

For SUMO fusions, I amplified Smt3 (SUMO) from yeast genomic DNA and cloned it into a pET-derived expression vector also as an N-terminal 6xHis fusion. The resulting construct is called pTB146 [PT7::H-SUMO]. It was cloned so that precise gene fusions to SUMO can be constructed with NO ADDED sequences. You can then make you SUMO-protein fusions and cleave off the SUMO portion without leaving any additional non-native amino acids on your protein.

Strains included

BL21(lDE3)/plysS/pTB145 - for overexpressing the SUMO protease

DH5a/pTB146 - cloning strain with pTB146 for making SUMO fusion constructs

How to construct a SUMO fusion

See the vector map for pTB146. Use the SapI (GCTCTTC) site for the 5' end restriction site if you want to clone your gene and make a fusion protein that does not add any non-native residues following SUMO cleavage. There are many choices for the 3' end restriction site. I found XhoI to be particularly good for double digests with SapI.

The last four codons of SUMO code for QIGG and fusions are cleaved by SUMO protease after the GG. Be careful designing the 5' end primers used to amplify your insert fragment. SapI cuts outside of its recognition sequence, which is nonpalindromic (see NEB catalog). Everything has to be in the right orientation and the correct overhangs have to be engineered. When you cut the pTB146 vector with SapI it will accept a 5'-ggt-3' overhang on your digested PCR product. Make sure you draw out the 5' end of your insert fragment to ensure that SapI cleavage will generate such an overhang. Basically, when I design my oligos I just add the sequence 5'-cggt-3' between the SapI site and the start of my favorite gene sequence (make sure the SapI site is in the proper orientation).

This is all very awkward to explain in text, but it should hopefully make sense when you draw it out.

*IMPORTANT NOTE* SUMO protease will not cleave if your protein starts with Pro, Leu, Lys, or Val as the first amino acid after the GlyGly cleavage site.

Purifying H-Ulp1(403-621)

Grow a 5ml overnight culture of BL21(lDE3)/plysS/pTB145 in LB-Amp(50ug/ml)-Cam(25ug/ml)-Glucose(0.1%) at 30-37oC

Add to 500ml of LB-Amp-Glucose(0.04%) and grow at 30oC to an OD600=0.5

Add IPTG to 0.84mM and continue growing for 3.5-4hrs

Pellet cells, wash with 100ml Column buffer, pellet again, resuspend in 20ml Column buffer (50mM NaPi (pH 8.0), 300mM NaCl, 10mM imidazole) and transfer to 2x50ml conical tubes (Corning/Falcon). Snap freeze in N2(l) or dry-ice/acetone and store at -80oC.